DESCRIPTION: The presentation of adhesive proteins or peptides to cells in matrices has been demonstrated as a promising approach in the modulation of biological activities for tissue engineering and regenerative medicine. There are two primary routes for protein presentation, covalent attachment or adsorption. While covalent presentation has emerged to have better protein activity and retention, both covalent and adsorption approaches deprive cells of their native ability to remodel their immediate surroundings. Remodeling is achieved by transient associations between full-length fibronectin and structural components such as collagen, heparin, and other fibronectin molecules. It is an important process in both homeostasis and wound healing. To our knowledge, no 3D biomaterials currently exist that allow cells to remodel or reorganize the extracellular matrix proteins present in the material. The goal of this application is to use tunable protein-protein affinity interactions to enable transient immobilization of fibronectin within a 3D biomaterial and assess the impact of this immobilization scheme on cell functions related to wound healing. This will be achieved through a combination of protein engineering and bioconjugation utilizing a Src Homology 3 (SH3) domain and its associated SH3 binding peptides, and implemented with fibronectin in a degradable polyethylene glycol (PEG) hydrogel. Our hypothesis, based on preliminary data, is that transient immobilization of fibronectin will allow its remodeling by cells and enhanced motility, proliferation, and secretion of molecules important for tissue repair and growth. Three specific aims will be completed: (1) Fabricate PEG hydrogels containing transiently immobilized fibronectin; (2) Determine cell activities affected by transient fibronectin immobilization and measure protein remodeling; (3) Characterize initial in vivo host response to hydrogels containing transiently immobilized fibronectin. Though fibronectin and PEG hydrogel will be used as the model protein and biomaterial, respectively, the method is general and can be applied to many proteins and synthetic or natural biomaterials. This work will create an innovative protein presentation method that allows transient and tunable immobilization of proteins in a biomaterial for wound healing applications. It will also allow us and others to study cell remodeling of proteins in a controlled system and measure the effects it has on a variety of cellular functions.